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. Author manuscript; available in PMC: 2011 Feb 8.
Published in final edited form as: Circ Cardiovasc Genet. 2008 Dec;1(2):147–152. doi: 10.1161/CIRCGENETICS.108.811638

Determination of Paraoxonase 1 Status Without the Use of Toxic Organophosphate Substrates

Rebecca J Richter 1, Gail P Jarvik 1, Clement E Furlong 1
PMCID: PMC3035628  NIHMSID: NIHMS192471  PMID: 20031556

Paraoxonase 1 (PON1) is a member of a tandem 3-gene family localized on human chromosome 7q21-22.1 High-density lipoprotein-associated PON12,3 and PON34,5 are synthesized primarily in the liver, whereas PON2 is ubiquitously expressed.1 PON1 was initially characterized and named for its ability to hydrolyze paraoxon, the toxic oxon metabolite of parathion.6 Although Aldridge6 proposed in 1953 that serum paraoxonase (POase) and arylesterase (AREase) activities were carried out by the same enzyme, controversy about 1 versus 2 enzymes persisted for many years, resulting in a reclassification of POase/AREase from EC 3.1.1.2 to EC 3.1.8.1 for PON1 as an example of an organophosphorus (OP) hydrolase.7 The controversy was finally settled when Sorenson et al8 demonstrated both activities in recombinant PON1. However, the revised nomenclature remains in place. Early studies of plasma PON1 found a large variability of POase activity among different species and in different tissues.6 Serum POase activity distribution studies in human populations revealed an activity polymorphism of high versus low POase activity. Studies on the polymorphic distribution of PON1 in human populations using a variety of different assays revealed bi or trimodal distributions of plasma POase activity (reviewed in Ref.9).

Our initial characterization of the human PON1 cDNA clones revealed 2 coding region polymorphisms Q192R and L55 M.10 Subsequently, it was shown that the Q192R polymorphism determined high versus low rates of paraoxon hydrolysis by the enzyme, with the PON1R192 alloform specifying high activity.11,12 After the demonstration that high-density lipoprotein-associated PON1 was implicated in reducing low-density lipoprotein13 and high-density lipoprotein14 oxidation, epidemiological studies were undertaken to explore the possible role of genetic variability of PON1 in cardiovascular disease15 (reviewed in Ref.16). Several meta-analyses of studies that examined only the association of PON1 genotypes with risk of vascular disease have been published in recent years. The first meta-analysis in 2001 by Mackness et al17 examined the 19 studies carried out up to that time as part of a study of PON1 status in 417 coronary heart disease subjects and 282 controls. A second meta-analysis examined 38 studies in addition to their own,18 whereas a third analyzed 43 previous studies.19 Unfortunately, the majority of the epidemiological studies examined only PON1 genotypes using DNA methodologies and ignored the large interindividual variability in plasma PON1 activity levels. Fundamental biochemical and physiological principles dictate that rates of detoxication or metabolism depend on the quantity of enzyme present. Thus, it is not surprising that many analyses examining disease or exposure risk using only single-nucleotide polymorphism (SNP) analysis and not enzyme activity levels have been inconclusive. Several of the most experienced investigators in PON1 research have pointed out the inadequacy of examining PON1 genotype alone as a risk factor for disease or exposure.15,20–27 We introduced the term PON1 status to include both plasma PON1 activity levels and PON1192 genotype.23 The few studies that have examined PON1 status have found that plasma PON1 activity level is indeed a risk factor for vascular disease,17,25,26,28–31 whereas there was no association observed with PON1 genotypes.17,25,26

The importance of plasma PON1 activity level in protecting against OP exposure has been clearly demonstrated in the mouse and genetically modified mouse model systems.23,32–36 Resistance to diazoxon exposure is modulated primarily by PON1 plasma activity level, whereas both PON1 activity level and PON1 genotype are important in modulating exposures to chlorpyrifos oxon, due to substrate-specific differences in catalytic efficiency between the PON1Q192 and the PON1R192 alloforms.34

The most convenient protocol for determining PON1 status—plasma activity levels as well as functional position 192 genotype—makes use of a 2-substrate assay, 2-dimensional enzyme activity plot that displays rates of diazoxonase activity versus POase activity under high salt conditions.24,27,37,38 The high salt conditions are used to separate the PON1192Q/R data points from the PON1192R/R data points. Unfortunately, this protocol involves the use of 2 highly toxic OP substrates. We report here a 2-substrate assay/analysis protocol that makes use of non-OP substrates and is convenient for general laboratory use. A third assay that measures rates of phenyl acetate (PA) hydrolysis (AREase activity) at low salt concentration reveals plasma PON1 activity levels for all 3 PON1192 genotypes. Factors are provided to allow the conversion of rates of hydrolysis of one substrate to another for each PON1192 phenotype.

Methods

Subjects

The plasma samples used for this study came from an institutional review board-approved project investigating the role of PON1 in vascular disease. Plasma samples were drawn into lithium-heparin tubes, and the cells were separated from the plasma by centrifugation for 15 minutes at 1800g.

4-(Chloromethyl)phenyl Acetate Assay

CMPA [4-(chloromethyl)phenyl acetate] was obtained from Sigma Chemical Co (St Louis, Mo). Rates of CMPA hydrolysis were determined in a SPECTRAmax PLUS Microplate Spectrophotometer (Molecular Devices, Sunnyvale, Calif) using ultraviolet transparent 96-well microplates from Costar (Cambridge, Mass.). Rates of hydrolysis were measured at 280 nm for 4 minutes at 25°C. Only initial linear rates were used for calculations, and results were normalized using the path-length correction software supplied by the system manufacturer. Replicate assays that varied by >10% were repeated. Plasma samples were diluted 1:40 in dilution buffer [20 mmol/L Tris-HCl (pH 8.0), 1.0 mmol/L CaCl2] and 20 μL was added per microplate well. The data points were run in triplicate. The substrate solution for CMPA determinations was 20 mmol/L Tris-HCl (pH 8.0), 1.0 mmol/L CaCl2 to which CMPA was added to a final concentration of 3 mmol/L. The substrate solution was shaken vigorously for 30 seconds in a screw-capped polypropylene tube before use. Substrate solution (200 μL) was added to initiate the assay. Activities were expressed in Units/mL, based on the molar extinction coefficient of 1.30 mmol/L–1cm–1 for the CMPA hydrolysis product, 4-(chloromethyl)phenol.

Arylesterase Assays (High Salt/No Salt)

Rates of PA hydrolysis were determined in the SPECTRAmax PLUS Microplate Spectrophotometer using ultraviolet transparent 96-well microplates from Costar. Rates of hydrolysis of PA were measured for 4 minutes at 270 nm, with only initial linear rates used for calculations, with results normalized using the path-length correction software provided by the manufacturer. Replicates that varied by >10% were repeated. AREase assays used plasma dilutions (in dilution buffer) of 1:40 for assays run at high salt concentration and 1:80 for assays run at low salt concentration. The assay used 20 μL of diluted plasma per well to which 200 μL of 3.26 mmol/L PA substrate was added in either high salt assay buffer, or no salt assay buffer. High salt PA buffer contained 2 mol/L NaCl, 20 mmol/L Tris-HCl (pH 8.0), 1.0 mmol/L CaCl2, and low salt assay buffer contained 20 mmol/L Tris-HCl (pH 8.0), 1.0 mmol/L CaCl2. Activities were expressed in Units/mL, based on the molar extinction coefficient of 1.31 mmol/L–1cm–1 for phenol.

Paraoxonase and Diazoxonase Assays

Plasma PON1 activities toward paraoxon (PO) and diazoxon (DZO) and were determined as described previously.24,38 Paraoxon and diazoxon were obtained from Chem Service (West Chester, Pa.). Rates of paraoxon and diazoxon hydrolysis were determined in the SPECTRA-max PLUS Microplate Spectrophotometer using either ultraviolet transparent 96-well microplates from Costar for UV diazoxonase readings (270 nm) or standard flat bottom 96-well microplates from Greiner One (Monroe, N.C.) for visible wavelength POase readings (405 nm). All assays were carried out in triplicate using a multi-channel pipette (Matrix, Hudson, N.H.). Outlier samples were reassayed. Rates of hydrolysis were measured for 4 minutes, with only initial linear rates used for calculations and results normalized using path-length correction. PO hydrolysis rates (POase) were expressed in Units/liter (U/L), based on the molar extinction coefficient of 18 mmol/L–1cm–1 for p-nitrophenol. DZO hydrolysis (DZOase) activities were expressed in Units/liter (U/L), based on the molar extinction coefficient of 3 mmol/L–1cm–1 for the diazoxon hydrolysis product, 2-isopropyl-4-methyl-6-hydroxypyrimidine.

Results

Identification and Characterization of Nontoxic Discriminatory Substrates

The aim of this study was to develop assays for the determination of PON1 status (plasma PON1 activity levels and functional position 192 genotype) that do not use the highly toxic organophosphate substrates paraoxon and diazoxon. More than 70 compounds with many assay conditions (variation of salt concentration and pH) were examined in attempts to find 2 substrates and assay conditions that would provide the same resolution of the PON1192 phenotypes as the toxic DZO/PO substrate pair.24 Of the many substrates and conditions tried, hydrolysis of PA at 2 mol/L salt and CMPA at low salt provided the best resolution of functional PON1192 phenotypes. The primary requirement for useful substrates is that the substrate and assay conditions reveal different rates of hydrolysis between the PON1Q192 and the PON1R192 alloforms.24

Figure 1 shows the structures of the 4 substrates used to determine PON1 status. The first step in the design of a spectrophotometric assay for substrate hydrolysis is to examine the spectra of the unhydrolyzed ester and the released alcohol. Aromatic alcohols in general provide useful spectral shifts on hydrolysis. Figure 2 shows the spectra of CMPA and 4-(chloromethyl)phenol. The wavelength of 280 nm was chosen for continuous monitoring of the hydrolysis of CMPA.

Figure 1.

Figure 1

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Structures of the substrates used for determining PON1 status.

Figure 2.

Figure 2

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Spectra of CMPA (●–●) and 4-(chloromethyl)phenol (○–○) and the difference spectrum (+–+).

In designing assays that best discriminate between the 2 PON1192 alloforms, it was necessary to find conditions where the kinetic properties of the 2 alloforms differed sufficiently to separate the 3 PON1192 phenotypes or “functional genotypes” (QQ, QR, and RR). Previous experience with PON1 assays has shown that either pH and/or salt conditions are most conveniently used to generate optimal assay conditions that will resolve the 3 PON1192 phenotypes. Our earlier work also showed that it is necessary to measure PON1 activity at or below pH 8.5 to avoid interference from the esterase activity of albumin, which catalyzes a higher rate of OP hydrolysis than PON1 at high pH values in plasma samples with low PON1 activity levels.9 A pH value of 8.0 proved to be optimal for measuring rates of hydrolysis of both PA and CMPA (data not shown).

The effects of varying NaCl concentration on rates of PA hydrolysis are shown in Figure 3A. As with diazoxon hydrolysis,24 PON1R192 was more sensitive to inhibition by NaCl than was PON1Q192. Because most of the currently used assays are run at 2 mol/L NaCl, and this level of salt provided a good differentiation of the activity of the 2 PON1192 alloforms, 2 mol/L NaCl was selected for optimizing the spread of the data points for the “y axis substrate” PA. Because we have previously shown that rates of PA hydrolysis in absence of NaCl may be used to compare levels of plasma PON1 across PON1192 genotypes, rates of PA hydrolysis were also determined in the absence of salt.

Figure 3.

Figure 3

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Rates of hydrolysis of (A) PA by PON1Q192 (○–○) and PON1R192 (Δ–Δ); and (B) CMPA by PON1Q192 (○–○) and PON1R192 (Δ–Δ) as a function of sodium chloride concentration.

Figure 3B shows the dependence of rates of hydrolysis of CMPA by the 2 PON1192 alloforms as a function of salt. Interestingly, PON1R192 had higher rates of CMPA hydrolysis than PON1Q192 in the absence of salt, indicating that it would be a useful “x axis substrate.” Figure 4A shows the substrate dependence of PA hydrolysis by PON1Q192 (Km=443 μmol/L), and Figure 4B by PON1R192 (Km=279 μmol/L) at pH 8.0 in the presence of 2 mol/L NaCl. Figure 4C shows the substrate dependence of rates of hydrolysis of CMPA for PON1Q192 (Km=341 μmol/L), and Figure 4D by PON1R192 (Km=454 μmol/L) at pH 8.0 in the absence of NaCl. Km values were determined from plots of substrate concentration/velocity versus substrate concentration.39

Figure 4.

Figure 4

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Substrate dependent rates of hydrolysis of (A) PA by PON1Q192, (B) PA by PON1R192, (C) CMPA by PON1Q192, and (D) CMPA by PON1R192. The assays for PA hydrolysis were run at high salt and those for CMPA at low salt as described in Methods section. Closed symbols indicate plots of velocity (v) versus substrate concentration [S]; open symbols, [S]/v versus [S].

Comparison of the 2 Protocols for Determining

PON1 Status

Figure 5 compares the population distribution of rates of hydrolysis of the substrate pair DZO/PO (Figure 5A) with those of the substrate pair PA/CMPA (Figure 5B) for 183 individuals (PON1 status). Both distributions clearly resolve the 3 functional PON1192 phenotypes (QQ, QR, and RR). These plots reveal not only the PON1192 alloform(s) present in an individual's plasma but also the relative levels of each individual's plasma PON1 activity within each PON1192 phenotype, data that is for most considerations much more relevant for estimating risk than the PON1 SNP data. All PON1192 genotypes were correctly inferred by both 2-substrate analyses as verified by polymerase chain reaction assays.12 We have reported previously, mutations discovered by discrepancies observed between the PON1 status determinations and polymerase chain reaction analyses.27

Figure 5.

Figure 5

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Comparison of the 2 protocols for determining PON1 status. A, Assays using the highly toxic OP substrates DZO and PO. B, Assays using the non-OP substrates PA and CMPA. (○) indicates PON1Q/Q192;(■), PON1Q/R192;(Δ), PON1R/R192.

Generation of Assay Conversion Factors

To facilitate comparison of data obtained with this new protocol for establishing PON1 status with data generated with the DZO/PO substrate pair, plots of rates of hydrolysis of a given substrate versus rates of a second substrate for each PON1192 phenotype were prepared (data not shown) to obtain the conversion factors shown in the Table. It was necessary to determine the conversion factors for each PON1192 pheno-type separately because the catalytic efficiency of substrate hydrolysis differs for each phenotype. Because the rates of PA hydrolysis at low salt are not affected by PON1192 phenotype (Figure 6), they can be used to compare PON1 plasma activity levels across genotypes.40 Factors for converting rates of PA hydrolysis at high salt to low salt values were also determined so that it would not be necessary to run 3 different substrate assays for a study of PON1 status and plasma PON1 level determination (Table). If a laboratory environment has high ambient temperatures, it may be necessary to generate temperature correction factors.

Table.

Factors for Converting Rates of Hydrolysis of Substrates

Phenotype Conversion Factors r2
QQ AREaseHS (U/mL)×185=DZOase (U/L) 0.81
QR AREaseHS (U/mL)×205=DZOase (U/L) 0.90
RR AREaseHS (U/mL)×236=DZOase (U/L) 0.88
QQ CMPAase (U/mL)×18.9=POase (U/L) 0.92
QR CMPAase (U/mL)×36.3=POase (U/L) 0.90
RR CMPAase (U/mL)×54.3=POase (U/L) 0.95
QQ AREaseHS (U/mL)×1.6 = AREaseLS (U/mL) 0.95
QR AREaseHS (U/mL)×2.0=AREaseLS (U/mL) 0.66
RR AREaseHS (U/mL)×3.5=AREaseLS (U/mL) 0.83
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r2 indicates correlation coefficient squared; AREaseHS, arylesterase at high salt; AREaseLS, arylesterase at low salt.

Figure 6.

Figure 6

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Rates of PA hydrolysis for each PON1192 phenotype/genotype at low salt concentration: PON1Q/Q192 (○–○), PON1Q/R192, (□–□) and PON1R/R192 (Δ–Δ). Horizontal bars represent the mean AREase activity values for each phenotype.

Discussion

The studies on the relationship of genetic variability of PON1 to risk of disease or exposure now number into the hundreds. Unfortunately, most of these studies have looked for association of PON1 SNPs with susceptibility and have ignored the more important factor, plasma PON1 activity levels.15–19,25,26,28–31 In our initial characterization of human PON1 cDNA sequences, we identified 2 coding region SNPs (L55 M and Q192R).10 It was subsequently shown that it was the Q192R polymorphism that determined the catalytic efficiency of PON1,11,12 with PON1R192 having approximately 9-times the catalytic efficiency for hydrolysis of paraoxon compared with PON1Q192.34 The effects of the Q192R polymorphism are substrate dependent, with PON1Q192 having higher activity against some of the nerve agents and PON1R192 having higher activity against PO and chlorpyrifos oxon.37 Both alloforms hydrolyze DZO34 and PA40 with approximately the same efficiency. Further research from our laboratory and 2 others examined the effects of 5 PON1 promoter region polymorphisms on plasma PON1 levels.41–44 The promoter region polymorphism that had the largest effect on PON1 activity levels was the C-108T polymorphism that occurs in an Sp1 transcription factor binding site.45 Homozygotes for PON1C-108 had on average twice the plasma level of PON1 activity compared with PON1T-108 homozygotes.42

There have been a number of reports linking low PON1 activity levels to the L55 M polymorphism with the PON1M55 allele being associated with low activity levels. However, most of this effect seems to be related to linkage disequilibrium of PON1M55 with the inefficient PON1T-108 allele.42 Leviev et al have reported that message levels46 and stability47 of the PON1M55 alloform may also contribute to the lower levels of PON1 activity associated with the PON1M55 genotype. AREase levels in a study of 1527 postmenopausal women reported by Roest et al48 were lower among PON1M55 homozygotes when compared with heterozygotes and PON1L55 homozygotes across all 3 PON1C-108T genotypes, lending support to an independent effect of the PON1M55 allele.

Of the 70 substrates tested, only PA (at high salt) and CMPA (in absence of salt) provided resolution of the 3 PON1 phenotypes comparable with that provided by the paraoxon/ diazoxon substrate pair. Another advantage of this substrate pair is that rates of hydrolysis can be determined at saturating substrate concentration. This was not feasible for diazoxon, where a nonsaturating concentration of 1 mmol/L was chosen for convenience and substrate solubility.24,38

Conclusions

The protocols described here will allow most laboratories to determine individuals’ PON1 status without the use of toxic substrates. The conversion factors presented here will also allow for the comparison of newly generated data with data reported from earlier studies. Again, it is important to note that epidemiological studies that examine only PON1 SNPs will be missing data on PON1 activity levels, which are more important than genotype in estimating an individual's risk of disease or exposure. For some exposures, genotype can also be important,34 but in no case are PON1 activity levels unimportant for estimating risk. Analysis of all ≈200 PON1 DNA polymorphisms will not provide the critical information generated by the 2-substrate PON1 status analysis protocol (functional PON1192 genotype and plasma activity levels).48

Sources of Funding

This work was supported by National Institutes of Environmental Health Sciences (ES09883) (Dr Furlong), (ES04696) (Dr Checkoway), (ES07033) (Dr Eaton), and the National Heart, Lung, and Blood Institute (RO1 HL67406 and HL074366) (Dr Jarvik).

Footnotes

Disclosures

None.

References

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